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Enzymolysis is a key bioconversion process of lignocellulosic biomass. The optimization of enzymolysis is important for its efficiency and accuracy. There is potential to solve the problem of low reducing sugar in the conversion of lignocellulose to bioethanol. In this study, mixed cellulases (cellulase and β-glucosidase) were used in the enzymolysis of acid-exploded poplar wood residues. The mixed enzymolysis process was optimized by response surface area test, and its kinetics model was established based on the Michaelis-Menten equation. The optimal parameters of the mixed enzymolysis were: initial, pH 5.2; temperature, 46 °C; and cellulase to β-glucosidase ratio, 1.62. These parameters resulted in enzymatic saccharification efficiency 1.3 times as high as that of the control (conducted with un-optimized parameters). The modeling revealed that there was a strong correlation (R2 = 0.97) between substrate concentration and reaction rate. Multiple simultaneous saccharification and cofermentation (MSSCF) developed in the laboratory was also employed to verify the optimal parameters. The mixed enzymolysis process carried out with the optimal parameters achieved an ethanol concentration of 30.09 ± 0.49 g/L, which was 1.64 times higher than that conducted with un-optimized parameters. The fermentation time was also reduced by 24 h. Overall, the optimization of mixed enzymolysis process could enhance the efficiency of lignocellulosic directional conversion to bioethanol.

Crude glycerol is largely generated as the main by-product of the biodiesel industry and is unprofitable for industrial application without costly purification. The direct bioconversion of crude glycerol into 1,3-propanediol (1,3-PDO) by microorganisms is a promising alternative for effective and economic utilization. In this study, Klebsiella pneumoniae 2e was newly isolated for the conversion of crude glycerol into 1,3-PDO. Batch fermentation analysis confirmed that crude glycerol and its main impurities had slight impacts on the growth, key enzyme activity, and 1,3-PDO production of K. pneumoniae 2e. The 1,3-PDO yield from crude glycerol by K. pneumoniae 2e reached 0.64 mol 1,3-PDO/mol glycerol, which was higher than that by most reported 1,3-PDO-producing Klebsiella strains. Genomic profiling revealed that K. pneumoniae 2e possesses 30 genes involved in glycerol anaerobic metabolism and 1,3-PDO biosynthesis. Quantitative real-time PCR analysis of these genes showed that the majority of the genes encoding the key enzymes for glycerol metabolism and 1,3-PDO biosynthesis were significantly upregulated during culture in crude glycerol relative to that in pure glycerol. Further comparative genomic analysis revealed a novel glycerol uptake facilitator protein in K. pneumoniae 2e and a higher number of stress response proteins than in other Klebsiella strains. This work confirms the adaptability of a newly isolated 1,3-PDO-producing strain, K. pneumoniae 2e, to crude glycerol and provides insights into the molecular mechanisms involved in its crude glycerol tolerance, which is valuable for industrial 1,3-PDO production from crude glycerol. IMPORTANCE The rapid development of the biodiesel industry has led to tremendous crude glycerol generation. Due to the presence of complex impurities, crude glycerol has low value for industry without costly purification. Obtaining novel microorganisms capable of direct and efficient bioconversion of crude glycerol to value-added products has great economic potential for industrial application. In this work, we characterized a newly isolated strain, Klebsiella pneumoniae 2e, with the capacity to efficiently produce 1,3-propanediol (1,3-PDO) from crude glycerol and demonstrated its adaptation to crude glycerol. Our work provides insights into the molecular mechanisms of K. pneumoniae 2e adaptation to crude glycerol and the expression patterns of its genes involved in 1,3-PDO biosynthesis, which will contribute to the development of industrial 1,3-PDO production from crude glycerol.

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